Integrins, extracellular matrix molecules, and cytoskeletal proteins contribute in complex fashion to cell migration and signaling. We are addressing the following specific questions: 1. What subcellular structures and signaling pathways are important for rapid cell migration? 2. How are the functions of integrins, the extracellular matrix, and the cytoskeleton integrated, and how is the regulatory crosstalk between them coordinated to produce normal cell migration? We are using a variety of cell and molecular biology approaches to address these questions, including biochemical analyses, fluorescent chimeras, and live-cell phase-contrast or confocal time-lapse microscopy. We have generated a variety of fluorescent molecular chimeras and mutants of cytoskeletal proteins as part of a long-term program to analyze their functions in integrin-mediated processes. We have been focusing particularly on the functions of integrins and associated extracellular and intracellular molecules in the mechanisms and spatial regulation of cell migration. We recently published work establishing the critical role of topography of an extracellular matrix substrate in regulating cell phenotype, including morphology, cytoskeletal organization, and speed of migration in J. Cell Biology (2009). A major uncertainty, however, involves the mechanisms by which a simple, nearly one-dimensional (1D) fibrillar matrix pattern can induce cells to migrate as rapidly and efficiently as their migration in a fibrillar 3D cell-derived matrix compared to their slower migration in regular 2D tissue culture. One difference involves dynamics at the leading edge of migrating cells: using high-speed imaging, we have been finding that the periodic protrusion-retraction cycles of the plasma membrane at the leading edge differ in 1D compared to 2D environments. In 1D, there is only a single slender adhesion, in contrast to the multiple adhesions at the broad leading edge of cells in 2D, and this adhesive configuration accelerates leading edge lamellipodial cycle frequency and results in a 10-fold increase in net protrusion rate. Besides its rapidity, a second notable feature of 1D migration is its dependence on myosin II-based contractility. Cells in both 1D and 3D environments show substantial deficits in cell migration after inhibition of myosin II by blebbistatin, yet cells migrating on 2D surfaces are resistant to inhibition and often migrate more rapidly as the cells lose focal adhesions. Although 1D and 3D cell adhesions initially assemble at the same rate as 2D focal adhesions, we find that 2D adhesions are much less stable: a substantial proportion of 1D and 3D adhesions remain stable for >1000 seconds, while nearly all 2D adhesions disassemble within that time. The dynamics of vinculin and paxillin are also slowed in 1D versus 2D adhesions. These findings are consistent with a greater coupling of these components of the clutch mechanism that drives cell migration. This enhanced stability of the single cell adhesion formed on long fibrillar patterns is associated with persistent directional migration. When cells on 1D lines are treated with blebbistatin to inhibit all myosin II functions, the characteristic long, continuous linear adhesion to the underlying matrix is disrupted into unstable, tiny spot-like nascent adhesions. Our findings suggest that myosin II functions to maintain firm adhesion to fibrillar matrix patterns with engagement of the molecular clutch to mediate rapid, directional migration. Our previous study in Nature Cell Biology (2007) suggested the existence of cross-regulation between myosin IIA and microtubule dynamics regulating cell migration. At a molecular level, however, it was not clear how cells could coordinate the opposing actions of actomyosin contractility, which stabilizes focal adhesions, versus microtubules that help disassemble them, and then how these processes might regulate cell migration. Our current studies indicate that fibroblasts balance levels of contractility and microtubule acetylation to modulate cell migration. We are searching for the molecular mechanisms of this putative balance that regulate rates of cell migration. These ongoing studies on the functions of integrins and associated intracellular and extracellular molecules in cell migration center upon our ability to image live-cell molecular dynamics of early cell protrusions and intracellular myosins and microtubules. All of these processes need to be analyzed in parallel in real time and in more physiological 1D and 3D matrix environments to be able to understand the mechanisms of in vivo cell migration. This combined knowledge should provide novel approaches to understanding, preventing, or ameliorating migratory processes that cells use in abnormal development and cancer. An in-depth understanding of exactly how cells move and interact with their matrix environment will also facilitate tissue engineering studies.